Structural basis for the synergy of 4 '- and 2 '-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-C alpha- and 4'-C beta-OMe, and 2'-F with 4'-C alpha-methyl modification. The alpha- and beta-epimers of 4'-C-OMe-uridine and the a- epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'alpha-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'beta-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F, 4'-C alpha-OMe-U, 2'-F, 4'-C beta-OMe-U, 2'-OMe, 4'-C alpha-OMe-U, 2'-OMe, 4'-C beta-OMe-U or 2'-F,4'-C alpha-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'beta-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F, 4'-C alpha-OMe-U or 2'-F, 4'-C beta-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'/4'-C-modifications.